Immunostimulatory and antioxidant activities of the selenized polysaccharide from edible Grifola frondosa

Abstract Grifola frondosa polysaccharide (GFP2) was extracted and purified by anion‐exchange chromatography. A selenized G. frondosa polysaccharide, SeGFP2, was modified in selenylation by nitric acid–sodium selenite (HNO3‐Na2SeO3) method. Structural features were investigated, and the lymphocyte proliferation and antioxidant activities were compared taking GFP2 as control. SeGFP2 with a molecular weight of 2.12 × 104 Da was composed of mannose, glucose, and galactose with a ratio of 3.5:11.8:1.0. A typical absorption of selenium ester was observed in SeGFP2 molecule. SeGFP2 was proposed as a branched polysaccharide, which consisted of 1,3‐D‐Glcp, 1,6‐D‐Glcp, 1,4,6‐D‐Galp, and 1,3,6‐D‐Manp. SeGFP2 showed a linear filamentous structure with some branches. SeGFP2 could significantly promote T‐ or B‐lymphocyte proliferation and the enhancement was higher than GFP2. The in vitro antioxidant activities of SeGFP2 were more potent than GFP2. These present data suggested that selenylation could significantly improve the lymphocyte proliferation and in vitro antioxidant activities of GFP2.

Se-polysaccharides have been widely used to prepare organic Se compounds, and attracted tremendous attention of researchers and consumers recently Zhang, Gao, et al., 2021).
Se-polysaccharides could exert the efficacy of both polysaccharide and Se, and the biological activity is usually higher than that of polysaccharide or Se (Zhang, Gao, et al., 2021). Generally, Se-polysaccharides from biotransformation method mainly exist in plants, mushrooms, and microorganisms. The quality of the Sepolysaccharides is influenced by both the area and season. The Se content and selenium translation rate of the Se-polysaccharides from biotransformation method are relatively lower than chemical selenylation, even in a high selenium area or liquid medium (Zhang, Lu, et al., 2016). Recently, it has been reported that the chemical selenylation of Se-polysaccharides involved nitric acid-sodium selenite (HNO 3 -Na 2 SeO 3 ), glacial acetic acid-selenous acid (CH 3 COOH-H 2 SeO 3 ), glacial acetic acid-sodium selenite (CH 3 COOH-Na 2 SeO 3 ), and selenium oxychloride (SeCl 2 O) method .
Grifola frondosa, an edible mushroom assigned to the Polyporaceae family, has been found to have diverse medicinal values. Due to the existence of bioactive polysaccharides, G. frondosa has become increasingly popular and widely cultivated in China (Klaus et al., 2015). These polysaccharides have been reported to possess potential biological effects, including immunostimulatory, antioxidant, antitumor, antidiabetic, and antihypertensive activities Meng et al., 2017). Bioactive polysaccharides extracted from the fruit bodies or mycelia of G. frondosa have attracted the most attention due to their diverse structure and potentially significant pharmacological activities.
In this study, the G. frondosa polysaccharides were extracted and purified by anion-exchange chromatography, and modified in selenylation by HNO 3 -Na 2 SeO 3 method for the first time. The lymphocyte proliferation and antioxidant activities of selenized G. frondosa polysaccharides (SeGFP2) were evaluated taking G. frondosa polysaccharides as control. The structural features of SeGFP2 were explored by Fourier transform-infrared (FT-IR) spectrometry, monosaccharide components analysis, methylation, gas chromatographymass spectrometry (GC-MS), high-performance size exclusion chromatography-multiangle laser light scattering-refractive index detector (HPSEC-MALLS-RI), Congo red spectrophotometric analysis, circular dichroism (CD), and atomic force microscope (AFM). Overall, this information would be helpful for the development of novel functional foods or drugs using the Se-polysaccharide as an ingredient.

| Extraction and purification of G. frondosa polysaccharide
The crude polysaccharides were extracted from the fruiting bodies of G. frondosa using a method reported before (Li et al., 2018).
Briefly, dry G. frondosa (40 g) was extracted with 1200 ml doubledistilled water at 100°C for 3 h, and the extraction process was repeated for three times. After centrifugation, the supernatants were combined and concentrated using a rotary evaporator. Then, four volumes of ethyl alcohol (EtOH) were added and the mixture was stored at 4°C overnight to precipitate polysaccharides.
The crude polysaccharides were purified by trichloroacetic acid method and column chromatography of DEAE-52 cellulose. The precipitates were redissolved in water and placed in an ice bath, followed by a slow addition of 15% trichloroacetic acid until the pH reached 2.0 ~ 3.0. After remaining for 4 h, the supernatants were collected, centrifuged, and adjusted pH to 7.0 with 1 M NaOH.
The solution was extensively dialyzed for 72 h (MWCO 3500 Da), and lyophilized to obtain the crude polysaccharides (GFP). GFP was redissolved and subjected to a DEAE-52 cellulose column (1.6 cm × 50 cm), followed by a stepwise elution using an increasing concentration of NaCl (0, 0.05, 0.10, 0.15, and 0.20 M) at a flow rate of 1.0 ml/min. Fractions were collected and the sugar profile was monitored using the phenol-sulfuric acid method. Fractions with the highest yield (0.05 M NaCl elution) were combined, concentrated, and lyophilized, generating the purified polysaccharide (GFP2).
Briefly, the purified GFP2 (30 mg) was dissolved in 0.7% HNO 3 and stirred at room temperature for 10 h. Na 2 SeO 3 of 24 mg and BaCl 2 of 40 mg were added and reacted at 70°C for 6 h. After the reaction, the mixture was cooled to room temperature and the pH was adjusted to 7.0 ~ 8.0. Na 2 SO 4 of 40 mg was added to remove the Ba 2+ . The supernatant was collected and dialyzed (MWCO 3000 Da) using distilled water until the reaction solution became colorless when detected by ascorbic acid method . The resulting solution was concentrated, precipitated with EtOH, and freeze dried to obtain selenized G. frondosa polysaccharides (SeGFP2).

| FT-IR spectroscopy
The FT-IR spectrum (4000-500 cm -1 ) was obtained using a NEXUS 670 FT-IR spectrophotometer. Two milligram of SeGFP2 was completely mixed with 200 mg of KBr and pressed into flakes. Singlebeam spectra were collected against that of the background reference and converted to the absorbance.

| Monosaccharide composition analysis
For GC analysis, SeGFP2 (5 mg) was hydrolyzed with 3 M H 2 SO 4 at 110°C for 8 h. After totally removing the excess H 2 SO 4 , the resultant monosaccharides were converted into alditol acetates as described before (Li et al., 2018), and then analyzed by GC.

| Methylation and GC-MS analysis
The glycosidic linkage analysis of SeGFP2 was carried out using the methylation method . Specifically, SeGFP2 (5.0 mg) was dissolved in anhydrous DMSO with a nitrogen inlet.
Dried NaOH (100 mg) was added and the mixture was stirred for 1 h. CH 3 I of 1.0 ml was added and the mixture was incubated in darkness for 4 h. The reaction mixture was extracted with chloroform, then the organic phase was washed with distilled water and dried under vacuum. After being methylated several times, the methylated SeGFP2 was confirmed by FT-IR. The methylated SeGFP2 was hydrolyzed with 85% formic acid at 100°C for 6 h and 2 M trifluoroacetic acid (TFA) at 100°C for 6 h, and then reduced with NaBH 4 and neutralized with acetic acid. The sample was acetylated by a procedure as mentioned in Section 2.6. Subsequently, the partially O-methylated alditol acetates (PMAAs) were detected by a GC-MS (6890N/5975B GC-MS, Agilent Co.) and the methylated SeGFP2 linkages were obtained by the retention time and fragmentation pattern. wyatt software (Wyatt Technology Co.) was used for the data acquisition and analysis.

| Colorimetric determination with Congo red
The helix coil transition or random coils conformation of SeGFP2 was determined by Congo red test. Usually, SeGFP2 (5 mg) was dissolved in water and then mixed with 80 μM Congo red dye. One M NaOH was dropwise added into the mixture to achieve 0 ~ 0.5 M final concentrations, and the absorbance (A) was recorded on an ultraviolet-visible spectrophotometer (UV-2450, Shimadzu Co.). The optical rotation of mixture alkaline solution without polysaccharides was used as the reference.

| Microscopic analysis
The atomic force microscopy (AFM) was employed to observe the molecular morphology of SeGFP2. SeGFP2 (10.0 µg/ml) was dispersed in water and filtered through a 0.45 μm syringe filter, and 10.0 µl of sample solution was deposited onto the freshly cleaved mica and then air dried for 4 ~ 8 h at room temperature. The sample was examined in the tapping mode with a Multimode 8 (Bruker) in air.

| Splenocyte proliferation assay
Lymphocyte proliferation was assessed by an MTT-based colorimetric assay. Balb/c mice (6 ~ 8 weeks old, 20 ± 2 g) were sacrificed via cervical dislocation. Spleens were aseptically removed and placed in cold RPMI-1640 medium under aseptic conditions, then gently homogenized, passed through a 40 μm nylon cell strainer to generate single-cell suspensions. After removal of erythrocytes from the cell mixture, the cells were washed twice and suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, adjusted to a final density of 5 × 10 6 cells/ml. Aliquots of 100 μl of splenocytes (5 × 10 6 cells/ml) were placed in a 96-well plate with or without ConA (10 μg/ml) or LPS (20 μg/ml). Samples of different concentrations (0, 25, 50, or 100 μg/ml) were added to each well and the plate was incubated at 37°C in a humidified 5% CO 2 incubator for 72 h.
Twenty microliters of MTT (5 mg/ml) was added per well and incubated for 4 h, followed by the addition of DMSO (150 μl/well).
The absorbance at 570 nm was measured using a microplate reader (BioTek Synergy H4).

| Antioxidant activity analysis
The in vitro antioxidant activities of SeGFP2 and GFP2 were evaluated using the free radical scavenging activities and ferrous ion-chelating abilities. The DPPH radical scavenging activity was measured according to the method described by . The ferrous ion-chelating ability was performed following the modified method described by Yuan et al. (2020). Briefly, the sam- where A B and A S separately represent the absorbance of blank and test sample. EDTA was co-assayed as a positive control.

| Statistical analysis
Data analysis was performed with SPSS software (version rel. 18.0, SPSS Inc.). Differences were considered statistically significant at p < .05.

| Extraction, purification, and general analysis of SeGFP2
A homogeneous polysaccharide fraction was purified by DEAE-52 column and named GFP2 (Li et al., 2018). The UV spectrum of GFP2 exhibited a decreasing absorbance similar to that of most polysaccharides, a negative response to the Bradford test and no absorption peaks at 260 or 280 nm, indicating the absence of nucleic acids and proteins.
The Se content of SeGFP2 was detected to be 445.39 μg/g.
Organic Se fortification of this mushroom source could help to alleviate Se deficiency in the population of China, in addition, making more Se-fortified food choices available. Similar Se content was also found in the selenized polysaccharide (SeASP 6 ) from Artemisia sphaerocephala after the HNO 3 -Na 2 SeO 3 synthesis method .
The alditol acetates of acid hydrolyzed SeGFP2 were analyzed by GC; in conclusion, it contained mannose, glucose, and galactose in a ratio of 3.5:11.8:1.0 (Figure 1). SeGFP2 was a heteropolysaccharide, in which D-glucose was the dominant constituent. A higher proportion of glucose was indicated in SeGFP2 compared with the previous reported study on G. frondosa polysaccharide, which consisted of glucose (64.4%), galactose (25.7%), and mannose (9.9%) (Xu et al., 2010).

| FT-IR spectra
The characteristic absorptions of SeGFP2 and GFP2 were performed using FT-IR spectra (4000 ~ 500 cm -1 ) (Figure 2). In the spectrum of GFP2, there were four characteristic absorption peaks at 3387.6 cm -1 , 2925. While most peaks were also shown in the spectrum of SeGFP2, two new absorption peaks appeared at 667.9 cm -1 and 1024.2 cm -1 , respectively, assigned to the Se-O-C stretching vibration (ν, 700 ~ 600 cm -1 ) and the O-Se-O stretching vibration (ν as , 1040 ~ 1010 cm -1 ) . This signified that Se had been combined to the polysaccharide molecule. The absorption band at 864.0 cm -1 was ascribed to β-type glycosidic linkages (Zhang, Zhou, et al., 2016). Previous studies indicated that active polysaccharides in those mushrooms appeared to have potential immunoregulatory activity, primarily due to the polysaccharides with β-glucan structures (Xu et al., 2012).

| Molecular weight and chain conformation
High-performance size exclusion chromatography-multiangle laser light scattering-refractive index detector was used as an efficient method to determine the molecular conformation and related parameters of the polysaccharide in dilute polymer solution. As shown in Figure 4, a single symmetrical peak was observed in the HPSEC chromatogram, and indicated that SeGFP2 was a homogeneous polysaccharide with the weight-average Mw of 2.12 × 10 4 Da. The polydispersity index M w /M n was 1.068, suggesting a polydisperse polymer in SeGFP2. The radius of gyration (R g ) is known as the distance between the mass center and the segment. The value of Z-average R z was determined to be 13.5 nm. For a given polymer solution, the gradient value (ν) may provide additional insights into macromolecule conformation and architecture. Usually, the ν values of 0.33, 0.50 ~ 0.60, and 1.0 separately exhibit the sphere, random coil, and rigid rod of the polymer (Zhao et al., 2014). The ν value of SeGFP2 was 0.40, which suggested that SeGFP2 molecules in an aqueous solution might be in a state between spheres and random coils.
Congo red test was performed to detect the triple helix or random coils structure of polysaccharide chains in an aqueous alkaline solution. Figure 5 shows the change of maximum absorbance (λ max ) of SeGFP2-Congo red complex at a NaOH concentration (0 ~ 0.5 M). Obviously, the addition of SeGFP2 to the Congo red solution did not cause any notable changes in λ max from 480 nm to 520 nm compared with that of Congo red alone, suggesting that SeGFP2 chains existed as random coils instead of helical structure. Furthermore, our results are consistent with the other bioactive polysaccharides, which also exhibited as random coils (Lavi et al., 2006).

| Molecular morphology
Generally, AFM is useful for observing the surface and topography of each sample (Kong et al., 2015). AFM images of SeGFP2 were provided in Figure 6. SeGFP2 appeared as worm-like chains and the molecular chains branched and entangled with each other at a concentration of 10.0 μg/ml. The height of all observed chains was around 0.3 ~ 8.1 nm, which is consistent with the thickness of multiple molecular chains (Liu et al., 2013). The molecular aggregation was ascribed to the -OH groups of SeGFP2, which provided inter/ intra-molecular interactions with each other or water molecule (Kong et al., 2015).

| Lymphocyte proliferation
Many fungal polysaccharides can activate T lymphocyte and B lymphocyte to show their effects on the immune system (Liu et al., 2017). Lymphocyte proliferation is the most direct indicator of immunoactivation. Usually, lymphocytes induced by ConA or LPS are, respectively, used to evaluate T-or B-lymphocyte activity (Liu et al., 2017). As shown in Figure 7a, the lymphocyte proliferation rate in SeGFP2 or GFP2 group was significantly (p < .05) higher than that of the control group. SeGFP2 groups at 25, 50, and 100 μg/ml were significantly (p < .01) higher than corresponding ConA control group (Figure 7b). Synergistic effect was observed between polysaccharide and LPS, especially at the medium and high concentrations with ConA or LPS, and the enhancement was higher than GFP2.
Immunostimulation itself is regarded as one of the important strategies to improve the host defense mechanism in humans as well as cancer patients. Various experiments proved that polysaccharides from mushrooms could enhance the host immune system by stimulating T cells, B cells, natural killer cells, and macrophage cells (Liu et al., 2017;Xu et al., 2012). Grifola frondosa polysaccharides were reported to show immunostimulatory activities, such as the improvement of RAW264.7 cells proliferation and the macrophageactivating capability (Meng et al., 2017). In fact, immunostimulatory activities of polysaccharides depend on the structural information such as monosaccharide constituent, glycosidic linkage, molecular weight, and function groups. It was reported that β-glucans from mushrooms, especially β-1,3-and β-1,6-linkages, were important for increasing cell immune activity (Liu et al., 2017;Xu et al., 2012). Sun et al. (2012) reported that a relatively low molecular weight of the polysaccharide was desired for its immunostimulatory activity.

F I G U R E 3
The total ion chromatograms from methylation analysis of SeGFP2 Se-polysaccharides were immune response regulators as reported in several studies . Haibo et al. (2016) reported that the selenylation modification of Chuanminshen violaceum polysaccharides (sCVPS) was obtained using HNO 3 -Na 2 SeO 3 method. The selenylation of CVPS could significantly increase the immunoregulatory activity both in vitro and in vivo, thus representing a powerful adjuvant for vaccine design. It was elucidated that Se alone could improve the abnormal levels of cytokines and oxidative damages in chicken spleen, thus ameliorating the injury induced by heat stress. The combination of Se and polysaccharides induced a higher immune function (Zhang, Gao, et al., 2021). Thus, these structural features may be responsible for the higher lymphocyte proliferation activity of SeGFP2. Further studies should be made to elucidate the immunostimulatory activity and its possible mechanism.

| Antioxidant activity
As illustrated in Figure 8a, the DPPH radical scavenging abili- The ABTS + radical has been widely used to measure the total antioxidant activity of single compounds or complex mixtures (Jeddou et al., 2016). As shown in Figure 8b, the effect of ABTS + rad- The chelating agents, which form bonds with metals, are effective as secondary antioxidants for the redox potential reduction, thus stabilizing the oxidized form of the metal ions (Yuan et al., 2020). Both SeGFP2 and GFP2 showed antioxidant activities, and the Fe 2+ -chelating ability was, respectively, 62.83% and 45.9% at a dose of 2000 μg/ml, lower than that of EDTA ( Figure 8c).
Basically, the chelating ability of SeGFP2 was a little superior to that of GFP2 under other six concentrations. As described by Yuan et al. (2020), the chelating ability of polysaccharides on Fe 2+ might affect the other radical scavenging activities to protect the organism against oxidative damage. Since Fe 2+ is the most effective pro-oxidant in food system, the high Fe 2+ -chelating abilities of polysaccharides from G. frondosa fruit bodies would be somewhat beneficial in the antioxidation.
It was reported that the antioxidant ability of polysaccharides was due to their hydrogen-donating effects. The element Se in SeGFP2 could activate the hydrogen atom of the anomeric carbon (Turło et al., 2010;Zhang, Lu, et al., 2016). The higher irritation ability of the group led the hydrogen atom-donating ability stronger.
This suggested that selenylation modification could enhance the in vitro antioxidant activity. In fact, a relatively low molecular weight of polysaccharides was highly desired for the antioxidant ability (Zhao et al., 2014). SeGFP2 with a M W of 2.12 × 10 4 Da exhibited a stronger antioxidant ability than Se-GFP-22 (4.13 × 10 6 Da), which was reported in our previous study . The antioxidant ability of the polysaccharides also strongly depended on the type of sugar monomers, the linkage pattern of the backbone, and the degree of branching.

| CON CLUS ION
In this study, G. frondosa polysaccharides were extracted and purified by anion-exchange chromatography, and modified in selenylation by HNO 3 -Na 2 SeO 3 method for the first time.

CO N FLI C T O F I NTE R E S T
There are no conflicts to declare.

E TH I C A L A PPROVA L
Animal experiments were performed in accordance with the code of ethics of the World Medical Association and approved by the Ethics Committee of Yangzhou University.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.